JP2002209596A - Method for producing l-amino acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the efficiency of L-amino acid production using a bacterium belonging to the genus Escherichia. SOLUTION: This method for producing the L-amino acid comprises culturing a bacterium belonging to the genus Escherichia and having an ability to produce the L-amino acid, in a culture medium containing fructose as a main carbon source, preferably a culture medium containing a carbon source comprising the fructose in an amount of >=30 wt.% and glucose in an amount of <=70 wt.%, thus producing and accumulating the L-amino acid in the culture medium, and then collecting the L-amino acid from the culture medium.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an L-fermentation method.
The present invention relates to a method for producing an amino acid. L-amino acids are widely used as raw materials for pharmaceuticals, seasonings, feeds, and the like.

[0002]

2. Description of the Related Art Heretofore, L-amino acids have been industrially produced by fermentation using coryneform bacteria belonging to the genus Brevibacterium or Corynebacterium. In recent years, methods for producing L-amino acids using bacteria belonging to the genus Escherichia such as Escherichia coli have also been developed. In addition, various techniques for increasing L-amino acid producing ability by genetic recombination techniques have been disclosed (Japanese Patent Laid-Open No. 57-7139).
7, U.S. Pat. No. 4,371,614).

[0003] By the way, it has been reported that when fructose is used as a carbon source when performing large-scale protein expression, the production of acetic acid is reduced and the yield of bacterial cells is increased (Bi).
otechnol. Prog., 15, 140-145 (1999)). However, the relationship between fructose and L-amino acid production is not known.

[0004]

As described above, although the productivity of L-amino acids has been considerably increased by breeding microorganisms and improving production methods, in order to respond to a further increase in demand in the future, L-amino acids are required. Further, there is a need for the development of a more inexpensive and efficient method for producing L-amino acids.

[0005] The present invention relates to L-type bacteria in the genus Escherichia.
-To provide a technique for improving the efficiency of amino acid production.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems. As a result, when fructose is used as a carbon source in a medium used for culturing Escherichia bacteria, L-amino acid production is reduced. It has been found that the performance is improved, and the present invention has been completed.

That is, the present invention provides the following: (1) Escherichia bacteria having L-amino acid-producing ability are cultured in a medium containing fructose as a main carbon source, and L-amino acids are produced and accumulated in the medium. And collecting L-amino acids from the medium. (2) The method for producing an L-amino acid according to (1), wherein the medium contains 30% by weight or more of fructose based on the total amount of the carbon source. (3) the medium is at least 30% by weight based on the total amount of the carbon source;
(1) The method for producing an L-amino acid according to (1), containing 95% by weight or less fructose. (4) The medium contains 30% by weight or more of fructose and 70% by weight or less of glucose based on the total amount of the carbon source.
(1) A method for producing an L-amino acid. (5) The Escherichia bacterium is Escherichia coli
The method for producing an L-amino acid according to any one of (1) to (4). (6) the L-amino acid is L-tryptophan
The method for producing an L-amino acid according to any one of (1) to (5).

[0008] As used herein, the term "L-amino acid-producing ability" refers to the ability to accumulate a significant amount of L-amino acid in a medium when a bacterium belonging to the genus Escherichia is cultured in the medium.
Alternatively, it refers to the ability to increase the L-amino acid content in cells. In the present invention, L-amino acids include L-tryptophan, L-phenylalanine, L-lysine and L-lysine.
Threonine, L-valine, L-leucine, L-isoleucine, L-homoserine, L-glutamic acid and the like.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION The bacterium used in the present invention is not particularly limited as long as it is a bacterium belonging to the genus Escherichia and has the ability to produce L-amino acids. Specific examples of bacteria belonging to the genus Escherichia include the book by Neidhardt et al. (Neidhardt,
FC et.al., Escherichia coli and Salmonella Typhim
urium, American Society for Microbiology, Washington
DC, 1208, table 1), and their derivatives derived from bacteria.

[0010] Escherichia coli having L-amino acid-producing ability may be a mutant strain or a genetically modified strain. The mutant strain has a mutation that increases the intracellular activity of an enzyme involved in L-amino acid biosynthesis, specifically, a mutation that increases the expression level of the enzyme, or a mutation that releases feedback inhibition. Mutant strains. The transgenic strain may be a strain in which the copy number of a gene encoding an enzyme involved in L-amino acid biosynthesis is increased, or a strain in which an expression control sequence is modified so that the expression level of the gene is increased. Or a strain into which a gene encoding an enzyme whose feedback inhibition has been released has been introduced.

The mutant strain is irradiated with ultraviolet light or N-methyl-
It can be obtained by treating a wild strain of the genus Escherichia or a derivative thereof with a mutagen such as N'-nitro-N-nitrosoguanidine (NTG) or nitrite, which is commonly used for mutagenesis.

In order to increase the copy number of a gene, a target gene and a vector that functions in a bacterium belonging to the genus Escherichia may be ligated to prepare a recombinant DNA, and the bacterium belonging to the genus Escherichia may be transformed with the recombinant DNA. Transformation was performed using DA
Morrison's method (Methods in Enzymology, 68, 326, 197)
9) Alternatively, treat recipient cells with calcium chloride
Methods to increase NA permeability (Mandel, M. and Higa, A., J. Mo
l., Biol., 53, 159 (1970)).
Examples of the vector include pUC19, pUC18, pUC118, pUC11
9, pBR322, pHSG299, pHSG298, pHSG399, pHSG398, RSF
1010, pMW119, pMW118, pMW219, pMW218, pSTV28, pSTV
29 and the like, and other phage vectors can also be used.

Increasing the copy number of a gene can also be achieved by allowing the target gene to exist in multiple copies on the chromosomal DNA of a bacterium belonging to the genus Escherichia. In order to introduce a target gene in multiple copies into the chromosomal DNA of a bacterium belonging to the genus Escherichia, homologous recombination is performed using a sequence present in multiple copies on the chromosomal DNA as a target. Sequences present in multiple copies on chromosomal DNA include repetitive DNA,
Inverted repeats at the end of the transposable element are available. Alternatively, as disclosed in Japanese Patent Application Laid-Open No. 2-109985, it is also possible to mount a target gene on a transposon, transfer it, and introduce multiple copies into chromosomal DNA.

[0014] The activity of the target enzyme can also be increased by replacing an expression control sequence such as a promoter of a gene encoding the target enzyme with a strong one (see JP-A-1-215280). For example, lac promoter, trp promoter, trc promoter, tac promoter, P _R promoter of lambda phage, P _L promoter, tet promoter, amyE promoter and so forth are known as strong promoters.

Hereinafter, L-tryptophan-producing bacteria as L-amino acid-producing bacteria belonging to the genus Escherichia will be described.
Breeding methods and specific strains are exemplified. Examples of enzymes involved in the biosynthesis of L-tryptophan include L-tryptophan biosynthesis 3-deoxy-D-arabino-heptulonic acid-7-phosphate synthase (see Japanese Patent Application Laid-Open No. 5-236947).
Transketolase (U.S. Pat. No. 5,906,925), anthranilate synthase (WO94 / 08031 (Tokuhyohei Hei 5-507693)
No.)), phosphoglycerate dehydrogenase (WO94 / 0
No. 8031). Among these enzymes, it is known that anthranilate synthase is feedback-inhibited by L-tryptophan and phosphoglycerate dehydrogenase is feedback-inhibited by L-serine.

Preferred as L-tryptophan-producing Escherichia bacteria used in the present invention are Escherichia bacteria having desensitized anthranilate synthase, desensitized phosphoglycerate dehydrogenase, or both. is there. A bacterium belonging to the genus Escherichia having such properties can be obtained, for example, by mutating an anthranilate synthase gene (trpE) and / or a phosphoglycerate dehydrogenase gene (serA) so as not to be subjected to feedback inhibition. The gene can be obtained by introducing the gene into Escherichia attribute bacteria. More specifically, such a bacterium belonging to the genus Escherichia includes, in Escherichia coli SV164 carrying a desensitized anthranilate synthase, a plasmid pGH5 (WO94) having a mutant type serA encoding a desensitized phosphoglycerate dehydrogenase. / 08031) can be mentioned.

Also, a bacterium belonging to the genus Escherichia, into which a recombinant DNA containing the tryptophan operon has been introduced, is suitable L.
-Tryptophan-producing bacteria. Specific examples include Escherichia coli into which a tryptophan operon containing a gene encoding a desensitized anthranilate synthase has been introduced (JP-A-57-71397, JP-A-62-244382,
U.S. Pat. No. 4,371,614).

Further, as an L-tryptophan-producing bacterium, a strain Escherichia coli AGX17 (pGX44) [NRRL] having a trait requiring L-phenylalanine and L-tyrosine is used.
B-12263], and AGX6 (pGX50) aroP (NRRL B-12264), which carries a plasmid pGX50 containing the tryptophan operon
(See U.S. Pat. No. 4,371,614).

In addition, the ability to produce L-tryptophan can be enhanced by increasing the ability to produce phosphoenolpyruvate in cells of the genus Escherichia having the ability to produce L-tryptophan (WO97 / 08333).

The above-mentioned enzyme genes or operons can be obtained according to a general gene isolation method well known to those skilled in the art. For example, primers are synthesized based on a known sequence, and PCR is performed using chromosomal DNA of a bacterium belonging to the genus Escherichia such as Escherichia coli K-12 as a template.
, It is possible to obtain the target gene.

Preparation of chromosomal DNA, preparation of chromosomal DNA library, hybridization, PCR, preparation of plasmid DNA, cleavage and ligation of DNA, transformation, setting of oligonucleotides used as primers, etc. How to introduce
Conventional methods well known to those skilled in the art can be employed. These methods are described in Sambrook, J., Fritsch, E.
F., and Maniatis, T., "Molecular Cloning A Laborato
ry Manual, Second Edition ", Cold Spring Harbor Lab
oratory Press, (1989).

Next, other L-amino acid producing bacteria will be exemplified below. Examples of L-phenylalanine-producing bacteria include Escherichia coli AJ 12604 (FERMBP-3579) (see European Patent Application Publication No. 488,424). Examples of L-lysine-producing Escherichia bacteria include mutants having resistance to L-lysine analogs. This L-lysine analog is such that it inhibits the growth of Escherichia bacteria, but its inhibition is such that if L-lysine coexists in the medium, it is totally or partially released. . For example, oxalidine, lysine hydroxamate, (S) -2-aminoethyl-L-cysteine (AE
C), γ-methyllysine, α-chlorocaprolactam and the like. Mutants having resistance to these lysine analogs can be obtained by subjecting a microorganism of the genus Escherichia to ordinary artificial mutation procedures. Specific examples of the strain used for producing L-lysine include Escherichia coli AJ1144.
2 (FERM BP-1543, NRRL B-1218
5; JP-A-56-18596 and U.S. Pat.
No. 170) and Escherichia coli VL611. The AJ11442 strain was acquired on May 1, 1981 by the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry of Japan.
05 Deposit No. 1-3-1 Higashi, Tsukuba, Ibaraki, Japan) under the accession number FERM P-5084.On October 29, 1987, the original deposit was transferred to an international deposit based on the Budapest Treaty. Deposited as FERM BP-1543. Aspartokinase of the above microorganisms is released from feedback inhibition by L-lysine.

Other examples include L-threonine-producing bacteria. This is because the inhibition of aspartokinase by L-lysine is generally released also from L-threonine-producing bacteria. Examples of L-threonine-producing bacteria of Escherichia coli include Escherichia coli MG442 (Gu
syatiner et al., Genetika (in Russian), 14, 947-956
(1978)).

In the above-mentioned L-lysine-producing bacterium, the L-lysine biosynthesis enzyme gene may be enhanced. Examples of such a gene include a gene encoding phosphoenolpyruvate carboxylase having a mutation that releases feedback inhibition by aspartic acid (see Japanese Patent Publication No. 7-83714). Specific examples of L-valine-producing Escherichia bacteria include Escherichia coli VL1970 (VKPM B-4411) (see European Patent Application Publication No. 519,113). Other examples include bacteria belonging to the genus Escherichia that carry an L-valine biosynthesis gene whose control mechanism has been substantially released. Such a bacterium belonging to the genus Escherichia is, for example, an ilvGMEDA operon, preferably an ilvGMEDA that does not express a threonine deaminase activity and has been attenuated.
It can be obtained by introducing an operon into a bacterium belonging to the genus Escherichia (see JP-A-8-47397).

The complete nucleotide sequence of the ilvGMEDA operon has been elucidated (Nucleic Acids Res. 15, 2137 (1
987)), so that colony hybridization using an oligonucleotide prepared based on this sequence or P
It can be obtained from chromosomal DNA of Escherichia coli by CR. D with ilvGMEDA operon
The introduction of the NA fragment into Escherichia coli can be performed by the method using the above-described plasmid, phage, and transposon.

Examples of L-leucine-producing bacteria belonging to the genus Escherichia include strains having β-2-thienylalanine resistance, strains having β-2-thienylalanine resistance and β-hydroxyleucine resistance (these are JP-B-62-34397). JP-A-8-70879) and strains having 4-azaleucine resistance or 5,5,5-trifluoroleucine resistance.

Examples of L-isoleucine-producing Escherichia bacteria include Escherichia coli KX141 (VKPM B-478).
1) (see EP-A-519,113).

Examples of L-threonine-producing Escherichia bacteria include Escherichia coli VKPM B-3996 (RIA 186).
7) (see US Pat. No. 5,175,107) and MG442 strain.

L-homoserine-producing Escherichia bacteria include strain C600 (Appleyard RK, Genetics, 39,
440-452 (1954)) and the NZ10 strain which is a Leu ⁺ revertant.

Examples of L-glutamic acid-producing bacteria belonging to the genus Escherichia include L-valine-resistant strains such as Escherichia coli B11 and Escherichia coli K-12 (ATC).
C10798), Escherichia coli B (ATCC11)
303), Escherichia coli W (ATCC 9637)
Is mentioned.

In the present invention, a medium containing fructose as a main carbon source is used for culturing a bacterium belonging to the genus Escherichia having L-amino acid producing ability. Using fructose as the primary carbon source improves the yield and production rate of L-amino acids relative to sugar.

The carbon source may be substantially only fructose, or may contain a carbon source other than fructose. The content of fructose is preferably at least 30% by weight based on the total amount of the carbon source, and
0 to 70% by weight is more preferred, and around 50% is particularly preferred. Other carbon sources include glucose, sucrose, maltose and the like. Of these, glucose is preferred. Specifically, as a carbon source used in the present invention, 30% by weight or more of fructose,
A mixture consisting of 0% by weight or less of glucose is exemplified.

The medium components other than the carbon source are ordinary medium components such as a nitrogen source, inorganic ions, and organic trace nutrients used as required. As the nitrogen source, inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia and the like can be used.

As inorganic ions, small amounts of potassium phosphate, magnesium sulfate, iron ions, manganese ions and the like are added. As organic trace nutrients, it is desirable to suitable amount necessary required substances or yeast extract, and the like, such as vitamin B _1.

The cultivation may be carried out under conditions according to the strain to be used. Specifically, the cultivation is preferably carried out under aerobic conditions for 16 to 72 hours, at a culturing temperature of 30 ° C. to 45 ° C. The medium pH is controlled between 5 and 7. In addition, an inorganic or organic acidic or alkaline substance, furthermore, ammonia gas or the like can be used for pH adjustment.

The collection of L-amino acids from the fermentation broth is performed by
Depending on the type of amino acid, the method can be carried out by appropriately combining ion exchange resin method, precipitation method and other known methods.

[0037]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples.

<1> Construction of L-tryptophan-producing bacterium of Escherichia coli In accordance with the contents described in WO94 / 08031 (Japanese Patent Application Laid-Open No. 7-507693).
A mutant gene encoding anthranilate synthase whose feedback inhibition has been released (hereinafter, also referred to as deinhibited AS) was introduced into a trpE-deficient strain Escherichia coli KB862 (DSM7196) to obtain Escherichia coli SV164 (trpE8). Was.
Plasmid pGH5 (WO) containing a gene encoding phosphoglycerate dehydrogenase (hereinafter also referred to as mutant PGD) in which feedback inhibition was released was added to this SV164 strain.
94/08031). SV164 / pGH5 has tryptophan and serine producing ability. Escherichia coli KB862 was named AJ13828 and was published on December 21, 2000.
On the other hand, the Institute of Biotechnology and Industrial Technology,
5-8566 International Deposit at 1-3-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, and has been assigned the accession number FERM BP-7405.

Hereinafter, a method for constructing SV164 / pGH5 will be described. (1) Screening of Mutant Gene Encoding Deinhibited AS and Incorporation of the Mutant Gene into Chromosome Using 5-methyltryptophan which is a tryptophan analog, a mutant strain retaining deinhibited AS was screened. E.coli K12 YMC9 (ATCC33927) was applied to the mirror method (Miller JH, 1972, Experiments in Molecula
r Genetics, Cold Spring Harbor Laboratory, Cold Spri
ng Harbor, NY: 125-129).
Mutation treatment was performed with -nitro-N-nitrosoguanidine (NG). That is, about 2 × 10 ⁹ cells of YMC9 were transferred to 50 μg / NG
The mixture was incubated at 37 ° C. for 30 minutes in 4 ml of 0.1 M sodium citrate buffer (pH 5.5) containing ml. After washing twice with 0.1 M sodium phosphate buffer (pH 7.0),
After shaking at 37 ° C overnight, the cells were cultured in LB. Subsequently, the culture solution was Na
The mixture was diluted to 10 ⁻³ , 10 ⁻⁴ , and 10 ⁻⁵ with 0.9% Cl, and 0.1 ml of the diluted solution was spread on a minimal medium plate containing 100 μg / ml of 5-methyltryptophan. The composition of the minimal medium is glucose 5 g / l, vitamin B1 5 mg / l, KH ₂ PO ₄ 3 g / l, KH ₂ PO _{4
12g / l, MgSO 4 · 7H} 2 O 0.3g / l, NaCl 0.1g / l, (NH 4) 2 SO 4 5
g / l, it is _{CaCl 2 · 2H 2 O 14.7mg /} l, FeSO 4 · 7H 2 O 2mg / l, Na3 citrate 1 g / l and agar 15 g / l.

After 24-48 hours at 37 ° C., 5-methyltryptophan-resistant clones were seeded on the agar medium.
In order to examine the properties of the obtained mutant, the Ki value of AS against L-tryptophan was measured (Bauerle R. et a
l., 1987, Methods in Enzymology Vol. 142: 366-38
6). As a result, the mutants could be classified into two classes. Class 1 mutants had feedback-resistant anthranilate synthase. The mutants of class 2 had a high Ki anthranilate synthase activity, although their Ki values did not change. The AS genes of these mutants were cloned and their nucleotide sequences were determined. Chromosome of each mutant
DNA is isolated, cut with restriction enzymes NheI and ClaI, and
The NheI / ClaI fragment of pBR322 (415
8 bp).

The ligation reaction was performed using E. coli KB 862 (trpE) (DSM
7196). Clones capable of growing on minimal medium without L-tryptophan were selected. Plasmids that complement the trpE mutation were all 5 kb NheI /
It contained a ClaI fragment. The 5 kb NheI / ClaI fragment contains trpE and trpD, about 0.8 kb upstream of trpE, and trpE.
It contained about 1 kb sequence downstream of D. Table 1 shows the difference in the amino acid sequence of the mutant AS encoded by the plasmid (pE0, pE5, pE6, pE8) carried by each mutant strain, and the Ki value.

[0042]

[Table 1] Table 1 酵素 Enzyme amino acid sequence Ki / mM ──野生 Wild type trpE NPTA LFHQ LCGD RPAT LLLE SADI DSKD DLKS (SEQ ID NO: 1) 0.01 trpE0 ------------------------------------- E- 0.1 trpE5 -S ----- -------------------------------- 3.0 trpE6 ---------------- --------- F ----------- E-> 15 tprE8 -S ---------------------- ------------- E- 15 ────────────────────────────────── ──

According to the sequence analysis of the class 2 mutant enzyme, the mutation was found to be trp in the operator region of the trp promoter.
It was also shown to be present in the DNA region encoding the leader peptide. Mutations termed ΔtrpL1 and ΔtrpL2 contain a 136 bp fragment in the DNA region encoding the leader peptide.
Alternatively, it had a deletion of 110 bp in size. In the sequence registered under accession number V00372 of the EMBL data bank, in the ΔtrpL1 mutation, the deletion includes the region from position 33 to position 168,
The ΔtrpL2 mutation includes the region at positions 11-120.

In order to highly express the gene encoding the deinhibited AS, the two mutant classes were combined. In class 2, the ΔtrpL1 mutation was used. Plasmid pΔt
A 1.6 kb NruI fragment with a ΔtrpL1 mutation was isolated from rpL and the corresponding Nr of plasmid pE0, pE5, pE6 or pE8.
Replaced with the uI fragment. The resulting plasmids were pIE0, pIE5,
Named pIE6 or pIE8 and used for chromosomal integration by homologous recombination.

An NheI / ClaI fragment of about 5 kb in size was isolated from each plasmid using low-melting-point agarose, and recD strain PD106 [ΔtrpLD102] was transformed while keeping the linear form. As a transformation method, Cohen et al., (1972) Pro
USA. 69: _2, 110-2114, CaCl2 method was used. Clones that were able to grow on minimal medium without L-tryptophan and were ampicillin sensitive, ie, free of plasmids, were selected. From each strain, P1 transduction (Miller JH, 1972, Experiments in
Molecular Genetics, Cold Spring Harbor, NY: 201
−205) to encode a de-inhibited AS and Δtrp
The mutant trpE gene having the L1 mutation was transferred into the KB862 strain, and selected on a minimal medium containing no tryptophan.
Obtained strains, PD103 (trpE0), KB862 (trpE5), SV164
(trpE8) and SV163 (trpE6).

(2) Production of the serA gene that decodes phosphoglycerate dehydrogenase sensitive to serine. From E. coli B strain (ATCC 23226), the serA gene encoding PGD was cloned on plasmid vector pUC18. The B strain was cultured in LB at 37 ° C. overnight.
Cells were harvested by centrifugation (4000 g) and purified by Ausubel et al., 198
7, 2.4.1−2.4.2, Current Protocols in Molecular Bi
Chromosomal DNA was prepared by the method described in ology, Greene Publishing Associates. Chromosomal DNA 10μg Sp
Cut with hI. Approximately 3 μg of the digest were ligated with 0.2 μg of similarly pph18-cut plasmid pUC18. In the ligation reaction, serA
Mutant PC1523 (CGSC No.5411) (CGSC: E. coli Genetic
Stock Center, Department of Biology 255 OML, Yale
University, Postbox 6666, New Haven, CT, USA) was transformed by the method of Cohen et al.

Transformants were plated on minimal medium without L-serine. The grown clone was serA from E. coli.
The gene was contained in a 3.5 kb SphI fragment. Wild type s
The sequence of the erA gene is described in WO94 / 08031. s
The recombinant vector having the erA gene was named pGC3.

Using the wild-type serA gene obtained as described above, a serA gene encoding a mutant PGD lacking the C-terminal amino acid was prepared. pGC3 was cut with SalI and KpnI, and the obtained fragments were separated by agarose gel electrophoresis. Sal of 2.0 kb size including the full length of serA gene
The I-KpnI fragment was purified from the gel. This fragment (0.2 μg), equimolar amounts of HindIII / SalI-cut pUC18, and a double-stranded oligonucleotide obtained by annealing an oligonucleotide having the nucleotide sequence shown in SEQ ID NOS: 2 and 3 were ligated. This oligonucleotide complements seven of the eight last C-terminal codons of the serA gene and introduces a stop codon TAA instead of the eighth codon. Therefore, the PGD encoded by this mutant serA gene is shortened by one C-terminal amino acid residue. The plasmid containing the mutant serA was designated as pGH5. The mutant PGD encoded by the gene has a Ki value for serine of 0.1 μM to 50 μM, and the feedback inhibition by serine is released.

<2> Production of L-tryptophan The above tryptophan-producing bacterium Escherichia coli SV164 /
pGH5 was placed in a 500 ml Erlenmeyer flask in 50 ml of LB medium (1% tryptone, 0.5% yeast extract,
0.5%) at 30 ° C. for 7-8 hours under shaking (150 rotations /
Min).

About 1 ml of the above preculture was inoculated into 300 ml of a seed medium having the following composition. The cells were cultured at 30 ° C. for 11 to 15 hours at 800 rpm using a 1 L small fermenter.

[0051] [Seed Medium Composition] glucose _{5g / L KH 2 PO 4 12g} / L (NH 4) 2 SO 4 0.1g / L MgSO 4 · 7H 2 O 0.3g / L CaCl 2 · 2H 2 O 15mg / L FeSO ₄・ 7H ₂ O 2mg / L Na ₂ Citrate ・ 2H ₂ O 1g / L Trace element solution 1mg / L L-phenylalanine 40mg / L L-tyrosine 40mg / L Vitamin B1 5mg / L Tetracycline 15mg / L (trace element solution _{) Na 3 MoO 4 0.15g / LH} 3 BO 3 2.5g / L CoCl 2 · 6H 2 O 0.7g / L CuSO 4 · 5H 2 O 0.25g / L MnCl 2 · 4H 2 O 1.5g / L ZnSO 4 · 7H ₂ O 0.3g / L

30 ml of the above seed culture was mixed with 30 parts having the following composition.
0 ml of the main culture medium was inoculated. Using a 1 L small fermenter, the cells were cultured at 30 ° C. under agitation at 800 rpm while passing 1 vvm of compressed air sterilized by a sterilizing filter. During the culture period, the temperature was kept at 31 ° C, and the pH was kept at 6.7 with ammonia gas.

[Main culture medium composition] Glucose 17.5 g / L KH ₂ PO ₄ 1.5 g / L (NH ₄ ) ₂ SO ₄ 5 g / L NaCl 0.5 g / L MgSO ₄ .7H ₂ O 0.3 g / L CaCl _2. 2H ₂ O 15mg / L FeSO ₄・ 7H ₂ O 75mg / L Na ₂ Citrate.2H ₂ O 1g / L Trace element solution 1mg / L L-phenylalanine 750mg / L L-tyrosine 750mg / L Vitamin B1 5mg / L Yeast Extract 2.5g / L Tryptone 2.5g / L tetracycline 20 mg / L (trace element _{solution) Na 3 MoO 4 0.15g / LH} 3 BO 3 2.5g / L CoCl 2 · 6H 2 O 0.7g / L CuSO 4 · 5H 2 O 0.25 _{g / L MnCl 2 · 4H 2} O 1.5g / L ZnSO 4 · 7H 2 O 0.3g / L

During the culture, 700 g / L of a sugar solution having the composition shown in Table 2
The sugar concentration in the small fermenter was adjusted to 5-20 g / L by pumping (W / V) (sterilized in an autoclave). After 48 hours of culture, the concentration of L-tryptophan in the medium was measured. Table 3 shows the yield to sugar and the production rate. Table 3 shows the ratio with G100 as 1.

[0055]

[Table 2]

[0056]

[Table 3]

As a result, it was found that when fructose was mixed with glucose, both the yield to sugar and the production rate were improved as compared with the case where a sugar solution containing 100% glucose was flowed down. In particular, the ratio of fructose to carbon
In the vicinity of%, the highest productivity was shown.

According to the present invention, in the production of L-amino acids such as L-tryptophan using a bacterium belonging to the genus Escherichia, the yield to sugar and / or the production rate can be improved.

[Sequence List] <110> Ajinomoto Co., Inc. <120> Method for producing L-amino acid <130> P-7719 <140> <141> 2001-01-19 <160> 3 <170> PatentIn Ver. 2.0 <210> 1 <211> 32 <212> PRT <213> Escherichia coli <400> 1 Asn Pro Thr Ala Leu Phe His Gln Leu Cys Gly Asp Arg Pro Ala Thr 1 5 10 15 Leu Leu Leu Glu Ser Ala Asp Ile Asp Ser Lys Asp Asp Leu Lys Ser 20 25 30 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 2 cattcgcgcc cgtctgctgt aata 24 <210 > 3 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: synthetic oligonucleotide <400> 3 ctaggtaagc gcgggcagac gacattattc ga 32

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (reference) (C12N 1/21 C12R 1:19) C12R 1:19) C12N 15/00 ZNAA F-term (reference) 4B024 AA01 AA05 AA10 BA71 BA75 DA06 EA04 GA11 4B064 AE03 AE08 AE34 CA02 CA19 CC03 CC24 CD09 DA10 4B065 AA26X AB01 BA18 BB15 CA17 CA41 CA43 CA44

Claims (6)

[Claims] 1. A bacterium belonging to the genus Escherichia capable of producing L-amino acids is cultured in a medium containing fructose as a main carbon source, L-amino acids are produced and accumulated in the medium, and L-amino acids are collected from the medium. A method for producing an L-amino acid. 2. The method for producing an L-amino acid according to claim 1, wherein the medium contains 30% by weight or more of fructose based on the total amount of the carbon source. 3. The medium according to claim 1, wherein the content of fructose is 30% by weight or more and 95% by weight or less based on the total amount of the carbon source.
A method for producing the L-amino acid according to the above. 4. The method for producing an L-amino acid according to claim 1, wherein the medium contains 30% by weight or more of fructose and 70% by weight or less of glucose based on the total amount of the carbon source. 5. The method for producing an L-amino acid according to claim 1, wherein the Escherichia bacterium is Escherichia coli. 6. The method for producing an L-amino acid according to claim 1, wherein the L-amino acid is L-tryptophan.